scholarly journals GhNHX3D, a Vacuolar-Localized Na+/H+ Antiporter, Positively Regulates Salt Response in Upland Cotton

2021 ◽  
Vol 22 (8) ◽  
pp. 4047
Author(s):  
Junping Feng ◽  
Wenyu Ma ◽  
Zongbin Ma ◽  
Zhongying Ren ◽  
Yang Zhou ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Upland Cotton ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Egfp Fusion Protein ◽  
Ion Contents ◽  
Green Fluorescent ◽  
Steady State Balance

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.

Ca2+ mobilization through dorsal root ganglion Ca2+-sensing receptor stably expressed in HEK293 cells

2007 ◽  
Vol 292 (5) ◽  
pp. C1895-C1905 ◽  
Author(s):  
Emmanuel M. Awumey ◽  
Allyn C. Howlett ◽  
James W. Putney ◽  
Debra I. Diz ◽  
Richard D. Bukoski
Keyword(s):  
Dorsal Root Ganglion ◽  
Fusion Protein ◽  
Dorsal Root ◽  
Fluorescent Protein ◽  
Hek293 Cells ◽  
Pcr Analysis ◽  
Hek 293 ◽  
Intracellular Ca ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

The rat dorsal root ganglion (DRG) Ca2+-sensing receptor (CaR) was stably expressed in-frame as an enhanced green fluorescent protein (EGFP) fusion protein in human embryonic kidney (HEK)293 cells, and is functionally linked to changes in intracellular Ca2+ concentration ([Ca2+]i). RT-PCR analysis indicated the presence of the message for the DRG CaR cDNA. Western blot analysis of membrane proteins showed a doublet of 168–175 and 185 kDa, consistent with immature and mature forms of the CaR.EGFP fusion protein, respectively. Increasing extracellular [Ca2+] ([Ca2+]e) from 0.5 to 1 mM resulted in increases in [Ca2+]i levels, which were blocked by 30 μM 2-aminoethyldiphenyl borate. [Ca2+]e-response studies indicate a Ca2+ sensitivity with an EC50 of 1.75 ± 0.10 mM. NPS R-467 and Gd3+ activated the CaR. When [Ca2+]e was successively raised from 0.25 to 4 mM, peak [Ca2+]i, attained with 0.5 mM, was reduced by ∼50%. Similar reductions were observed with repeated applications of 10 mM Ca2+, 1 and 10 μM NPS R-467, or 50 and 100 μM Gd3+, indicating desensitization of the response. Furthermore, Ca2+ mobilization increased phosphorylated protein kinase C (PKC)α levels in the cells. However, the PKC activator, phorbol myristate acetate did not inhibit CaR-mediated Ca2+ signaling. Rather, a spectrum of PKC inhibitors partially reduced peak responses to Cae2+. Treatment of cells with 100 nM PMA for 24 h, to downregulate PKC, reduced [Ca2+]i transients by 49.9 ± 5.2% (at 1 mM Ca2+) and 40.5 ± 6.5% (at 2 mM Ca2+), compared with controls. The findings suggest involvement of PKC in the pathway for Ca2+ mobilization following CaR activation.

scholarly journals The Recretohalophyte Tamarix TrSOS1 Gene Confers Enhanced Salt Tolerance to Transgenic Hairy Root Composite Cotton Seedlings Exhibiting Virus-Induced Gene Silencing of GhSOS1

2019 ◽  
Vol 20 (12) ◽  
pp. 2930 ◽  
Author(s):  
Benning Che ◽  
Cong Cheng ◽  
Jiajia Fang ◽  
Yongmei Liu ◽  
Li Jiang ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Gene Silencing ◽  
Hairy Root ◽  
Virus Induced Gene Silencing ◽  
Yeast Mutant ◽  
Transgenic Hairy Root ◽  
Cotton Plants ◽  
Stems And Leaves ◽  
Root Vigor

The salt overly sensitive 1 (SOS1) gene encodes the plasma membrane Na+/H+ antiporter, SOS1, that is mainly responsible for extruding Na+ from the cytoplasm and reducing the Na+ content in plants under salt stress and is considered a vital determinant in conferring salt tolerance to the plant. However, studies on the salt tolerance function of the TrSOS1 gene of recretohalophytes, such as Tamarix, are limited. In this work, the effects of salt stress on cotton seedlings transformed with tobacco-rattle-virus-based virus-induced gene silencing (VIGS) of the endogenous GhSOS1 gene, or Agrobacterium rhizogenes strain K599-mediated TrSOS1-transgenic hairy root composite cotton plants exhibiting VIGS of GhSOS1 were first investigated. Then, with Arabidopsis thaliana AtSOS1 as a reference, differences in the complementation effect of TrSOS1 or GhSOS1 in a yeast mutant were compared under salt treatment. Results showed that compared to empty-vector-transformed plants, GhSOS1-VIGS-transformed cotton plants were more sensitive to salt stress and had reduced growth, insufficient root vigor, and increased Na+ content and Na+/K+ ratio in roots, stems, and leaves. Overexpression of TrSOS1 enhanced the salt tolerance of hairy root composite cotton seedlings exhibiting GhSOS1-VIGS by maintaining higher root vigor and leaf relative water content (RWC), and lower Na+ content and Na+/K+ ratio in roots, stems, and leaves. Transformations of TrSOS1, GhSOS1, or AtSOS1 into yeast NHA1 (Na+/H+ antiporter 1) mutant reduced cellular Na+ content and Na+/K+ ratio, increased K+ level under salt stress, and had good growth complementation in saline conditions. In particular, the ability of TrSOS1 or GhSOS1 to complement the yeast mutant was better than that of AtSOS1. This may indicate that TrSOS1 is an effective substitute and confers enhanced salt tolerance to transgenic hairy root composite cotton seedlings, and even the SOS1 gene from salt-tolerant Tamarix or cotton may have higher efficiency than salt-sensitive Arabidopsis in regulating Na+ efflux, maintaining Na+ and K+ homeostasis, and therefore contributing to stronger salt tolerance.

scholarly journals Cell Death of Nicotiana benthamiana Is Induced by Secreted Protein NIS1 of Colletotrichum orbiculare and Is Suppressed by a Homologue of CgDN3

2012 ◽  
Vol 25 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Kae Yoshino ◽  
Hiroki Irieda ◽  
Fumie Sugimoto ◽  
Hirofumi Yoshioka ◽  
Tetsuro Okuno ◽  
...  
Keyword(s):  
Cell Death ◽  
Nicotiana Benthamiana ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Screening ◽  
Secreted Protein ◽  
Virus Induced Gene Silencing ◽  
Colletotrichum Orbiculare ◽  
Green Fluorescent

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein–based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Development of a Keratinocyte-Based Screening Model for Antipsoriatic Drugs Using Green Fluorescent Protein Under the Control of an Endogenous Promoter

2002 ◽  
Vol 7 (4) ◽  
pp. 325-332 ◽  
Author(s):  
Arno Pol ◽  
Fred Van Ruissen ◽  
Joost Schalkwijk
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Cell Line ◽  
Reporter Gene ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Endogenous Promoter ◽  
Keratinocyte Cell ◽  
Egfp Fusion Protein ◽  
Green Fluorescent

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-ca resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

scholarly journals Heterogeneity ofAspergillus nigerMicrocolonies in Liquid Shaken Cultures

2010 ◽  
Vol 77 (4) ◽  
pp. 1263-1267 ◽  
Author(s):  
Charissa de Bekker ◽  
G. Jerre van Veluw ◽  
Arman Vinck ◽  
L. Ad Wiebenga ◽  
Han A. B. Wösten
Keyword(s):  
Protein Production ◽  
Fluorescent Protein ◽  
Laser Microdissection ◽  
Pcr Analysis ◽  
Complex Object ◽  
Ferulic Acid Esterase ◽  
Mrna Accumulation ◽  
Esterase Gene ◽  
Green Fluorescent ◽  
Two Populations

ABSTRACTThe fungusAspergillus nigerforms (sub)millimeter microcolonies within a liquid shaken culture. Here, we show that such microcolonies are heterogeneous with respect to size and gene expression. Microcolonies of strains expressing green fluorescent protein (GFP) from the promoter of the glucoamlyase geneglaAor the ferulic acid esterase genefaeAwere sorted on the basis of diameter and fluorescence using the Complex Object Parametric Analyzer and Sorter (COPAS) technology. Statistical analysis revealed that the liquid shaken culture consisted of two populations of microcolonies that differ by 90 μm in diameter. The population of small microcolonies of strains expressing GFP from theglaAorfaeApromoter comprised 39% and 25% of the culture, respectively. Two populations of microcolonies could also be distinguished when the expression of GFP in these strains was analyzed. The population expressing a low level of GFP consisted of 68% and 44% of the culture, respectively. We also show that mRNA accumulation is heterogeneous within microcolonies ofA. niger. Central and peripheral parts of the mycelium were isolated with laser microdissection and pressure catapulting (LMPC), and RNA from these samples was used for quantitative PCR analysis. This analysis showed that the RNA content per hypha was about 45 times higher at the periphery than in the center of the microcolony. Our data imply that the protein production ofA. nigercan be improved in industrial fermentations by reducing the heterogeneity within the culture.

scholarly journals Generation of New Salt-Tolerant Wheat Lines and Transcriptomic Exploration of the Responsive Genes to Ethylene and Salt Stress

2020 ◽  
Author(s):  
Qian Ma ◽  
Huajian Zhou ◽  
Xinying Sui ◽  
Chunxue Su ◽  
Yanchong Yu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Agricultural Land ◽  
Carotenoid Biosynthesis ◽  
Pcr Analysis ◽  
Salt Tolerant ◽  
Ethylene Response ◽  
The World ◽  
Antenna Proteins ◽  
Wheat Lines

Abstract Background: Wheat (Triticum aestivum L.) is a staple crop in the world, but is only moderately salt tolerant. However, salt stress affects one-fifth of irrigated agricultural land in the world, it is of great importance to cultivate salt-tolerant varieties to improve the global wheat production. Results: In this study, over 90,000 wheat seeds of cultivar ‘Luyuan502’ were mutated by EMS, and 2000 salt-tolerant lines were harvested from salinized field. By analysis of ethylene sensitivity, salt related physiological factors, and preliminary crop yield, 12 salt-tolerant wheat lines with high production were selected among the crop plants. Transcriptome analysis indicated that a large number of the transcripts levels were significantly altered, mainly based on antenna proteins involved in photosynthesis, biosynthesis of secondary metabolites, cyanoamino acid metabolism, carotenoid biosynthesis, thiamine metabolism, and cutin, suberine and wax biosynthesis pathways including CABs, PERs/PODs, BGLUs, CYP707s, and ZEPs. qRT-PCR analysis revealed that the expressions of salt-related genes in the wheat lines were mostly higher than the wild type, and salt stress can significantly increase the expression levels of the ethylene-related genes in the wheat lines. Based on transcriptomic data, nine novel wheat ERFs were identified and analyzed, and it is suggested that they may play important roles in mediation of ethylene response and salt tolerance.Conclusion: Salt-tolerant wheat mutant lines with ethylene insensitivity were obtained from screen of a wheat EMS-mutagenized pool. Transcriptome data showed that the mutant plants exhibit significant alterations in the antenna proteins involved in various biological processes. Expression analysis suggests that ERFs may mediate ethylene response and salt tolerance of the wheat lines.

scholarly journals Application of RNA Interference Technology to Acroporid Juvenile Corals

2021 ◽  
Vol 8 ◽  
Author(s):  
Ikuko Yuyama ◽  
Tomihiko Higuchi ◽  
Michio Hidaka
Keyword(s):  
Gene Expression ◽  
Rna Interference ◽  
Stress Response ◽  
Large Scale ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Pcr Analysis ◽  
Genes Encoding ◽  
Green Fluorescent

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

scholarly journals Overexpression of Grapevine VvIAA18 Gene Enhanced Salt Tolerance in Tobacco

2020 ◽  
Vol 21 (4) ◽  
pp. 1323 ◽  
Author(s):  
Wei Li ◽  
Changxi Dang ◽  
Yuxiu Ye ◽  
Zunxin Wang ◽  
Laibao Hu ◽  
...  
Keyword(s):  
Salt Stress ◽  
Salt Tolerance ◽  
Transgenic Plants ◽  
Transgenic Tobacco ◽  
Transcriptional Activation ◽  
Late Embryogenesis Abundant Protein ◽  
Pcr Analysis ◽  
Vitis Vinifera L ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants

In plants, auxin/indoleacetic acid (Aux/IAA) proteins are transcriptional regulators that regulate developmental process and responses to phytohormones and stress treatments. However, the regulatory functions of the Vitis vinifera L. (grapevine) Aux/IAA transcription factor gene VvIAA18 have not been reported. In this study, the VvIAA18 gene was successfully cloned from grapevine. Subcellular localization analysis in onion epidermal cells indicated that VvIAA18 was localized to the nucleus. Expression analysis in yeast showed that the full length of VvIAA18 exhibited transcriptional activation. Salt tolerance in transgenic tobacco plants and Escherichia. coli was significantly enhanced by VvIAA18 overexpression. Real-time quantitative PCR analysis showed that overexpression of VvIAA18 up-regulated the salt stress-responsive genes, including pyrroline-5-carboxylate synthase (NtP5CS), late embryogenesis abundant protein (NtLEA5), superoxide dismutase (NtSOD), and peroxidase (NtPOD) genes, under salt stress. Enzymatic analyses found that the transgenic plants had higher SOD and POD activities under salt stress. Meanwhile, component analysis showed that the content of proline in transgenic plants increased significantly, while the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) decreased significantly. Based on the above results, the VvIAA18 gene is related to improving the salt tolerance of transgenic tobacco plants. The VvIAA18 gene has the potential to be applied to enhance plant tolerance to abiotic stress.

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